Curcumin could suppress the growth of colon cancer cells in the mouse model. This anti-tumor activity of curcumin was exerted by inhibiting cell proliferation rather than promoting cell apoptosis. Further study suggested that curcumin inhibited cell proliferation by suppressing the Wnt/β-catenin pathway. MiR-130a was down-regulated by curcumin treatment, and overexpressing miR-130a could abolish the anti-tumor activity of curcumin. Our study confirms that curcumin is able to inhibit colon cancer by suppressing the Wnt/β-catenin pathways via miR-130a. MiR-130a may serve as a new target of curcumin for CRC treatment.Curcumin exhibits anti-tumor effects in several cancers, including colorectal carcinoma (CRC), but the detailed mechanisms are still unclear. Here we studied the mechanisms underlying the anti-tumor effect of curcumin in colon cancer cells. SW480 cells were injected into mice to establish the xenograft tumor model, followed by evaluation of survival rate with the treatment of curcumin. The expression levels of β-catenin, Axin and TCF4 were measured in the SW480 cells in the absence or presence of curcumin. Moreover, miRNAs related to the curcumin treatment were also detected  Colorectal carcinoma (CRC) is the second most common malignancy in China . AdvanceThe Wnt pathway is categorized as canonical and non-canonical signaling pathways, the latter of which is β-catenin independent . On the Curcumin is the major yellow pigment and spice in turmeric and curry, and is a powerful anti-cancer agent . Studiesin vitro and in vivo, supporting curcumin as a chemotherapy candidate for colon cancer. The anti-tumor activity of curcumin was demonstrated by repressing the Wnt/β-catenin pathway and inhibiting the proliferation of colon cancer cells. This process was regulated by repressing the expression of microRNA (miR)-130a, and overexpressing miR-130a could completely abolish the curcumin-induced anti-tumor activity in colon cancer.In this study, we found that curcumin could suppress colon cancer both Dulbecco’s Modified Eagle Medium (DMEM) was bought from GIBCO , and the fetal calf serum (FCS) was purchased from HyClone Laboratories . Anti-human β-catenin, Axin, TCF4, GAPDH antibodies and curcumin were purchased from Sigma–Aldrich . Cell counting kit-8 was purchased from Dojindo Laboratories .TM miRNA Isolation Kit . All primers for detecting miRNA expression were designed and synthesized by Genscript Co. Ltd.  using the mirVanaTM qRT-PCR Primer Sets. U6 was used as internal control to normalize the expression levels of each miRNA. The fold change in miRNA expression was determined by comparative CT method.Total RNA was isolated with a mirVanaThe colon cancer cell line SW480 was obtained from American Type Culture Collection  and cultured in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin (Sigma–Aldrich).3). Then the cells were washed three times with Annexin V binding buffer. After centrifugation, the supernatant was discarded. Cells were incubated for 30 min at 4°C with FITC-Annexin V according to standard procedure. PI was added before testing. Fluorescence was measured using a FACSCalibur , and data were analyzed by the Flowjo Software .Cells with different treatments were washed twice in FACS medium , 250 mM NaCl, 0.5% NP-40, 3 mM EDTA, and 1.5 mM EGTA with 10 μg/ml Aprotinin, 10 μg/ml leupeptin, 1 mM DTT, 1 mM PNPP and 0.1 mM Na3VO4 as protease and phosphatase inhibitor. After centrifugation, cell lysates (100 μg/lane) were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes . The membranes were blocked for 1 h in TBST  containing 5% nonfat dried milk, and then incubated with appropriate antibodies diluted in TBST containing 5% nonfat dried milk at 4°C overnight. HRP-conjugated secondary antibodies were from Sigma. Protein bands were detected by the Immobilon Western Chemiluminescent HRP Substrate  and images were taken by FluorChem FC2 System .1 × 10The cell proliferation was detected by cell counting or by Cell Counting Kit (CCK)-8 according to the manufacturer’s instructions.The 5-bromo-2′-deoxyuridine (BrdU)  was incorporated into cellular DNA during cell proliferation. PE-conjugated BrdU mouse monoclonal antibody  was used to label the BrdU. After removing labeling medium, cells were fixed and DNA was denatured with fixing/denaturing solution. FACS was used to detect the incorporated BrdU by the PE channel.5 SW480 cells suspended in 1 ml DMEM were injected by axillary inoculation. Curcumin was injected by intraperitoneal (i.p.) injection at the dose of 200 mg/kg for 5 days after tumor generation. Mice were monitored daily to record the survival rate.6-week-old female nude mice were provided by Animal Center in Nanjing Medical University. All protocols, described below, were approved by the Animal Care and Use Committee at Wuxi People’s Hospital Affiliated to Nanjing Medical University. 1 × 10t-test was to examine the significance between two groups. P-values (p) were indicated in each figure.Results were expressed as mean ± SD. The one-way analysis of variance (ANOVA) was used to determine significant differences between the means of three or more independent groups, and the Student’s in vivo animal model by subcutaneous injection. The mice with the tumor receiving intraperitoneal injection of curcumin showed prolonged life span compare to the control group receiving vehicle injection . Meanwhile, the tumors in the curcumin group grew slower than the control group . These data indicated that curcumin exhibited anti-tumor activity in the in vivo mouse model generated by the SW480 cell line.Since curcumin was reported to have anti-tumor effects on colon cancer, we first used colon cancer cell line SW480 to generate the Figure 2A), which indicated that curcumin inhibited cell proliferation in vitro. Consistent with cell counting assay, results from the CCK-8 kit for determining the viability of the colon cancer cells also indicated that the curcumin-treated cells showed the lowest viability . Next, we tested the apoptosis of SW480 cells with different treatments. Percentages of the Annexin V-positive apoptotic cells with curcumin treatment did not display any difference compared to the control cells . These results indicated that curcumin suppressed colon cancer cells by inhibiting proliferation rather than promoting apoptosis. It is worth noting that similar results could be observed in another colon cancer cell line HCT116 . Since Wnt/β-catenin pathways have many negative regulators, such as Nkd2, we assessed the mRNA level of Nkd2 by RT-qPCR, and found that Nkd2 was upregulated by curcumin treatment . We then proceeded to assess the Wnt target gene TCF4. Although TCF is a transcription factor in the Wnt/β-catenin signaling, it is also a Wnt target gene. Consistent with our observations on Nkd2 and β-catenin, TCF4 was also down-regulated by curcumin treatment. The above results demonstrated that curcumin inhibited colon cancer cell proliferation through suppressing the Wnt/β-catenin signaling pathways.The Wnt/β-catenin pathways play an important role in colon cancer by promoting cell proliferation. Therefore, we asked whether the anti-tumor activity of curcumin was mediated through Wnt/β-catenin signaling. Western bolt analysis showed that the expression level of β-catenin was decreased after curcumin treatment, whereas the expression level of Axin was unchanged . MiR-21 has been reported to be involved in colon cancer, whereas the function of miR-130a is not clear. Therefore, we overexpressed miR-130a in SW480 cells and treated the cells with curcumin, followed by determining cell proliferation and β-catenin expression level. Interestingly, in the presence of miR-130a, cell proliferation was restored to similar level as the control group , and β-catenin level was also returned to normal level . These results indicated that curcumin suppressed the Wnt/β-catenin pathway via inhibiting miR-130a.In order to reveal the mechanism underlying curcumin regulation on the Wnt/β-catenin signaling, we tested several miRNAs reported to be upregulated in CRC. q-PCR showed that with curcumin treatment, miR-21 and miR-130a were down-regulated . Our results showed that miR-130a could repress the expression of Nkd2 in both mRNA and protein levels . We also predicted the targets of miR-130a through online tool1, among which LPR6 was identified to be related to the Wnt pathway. Therefore, we tested the level of LPR6 after miR-130a overexpression in the absence or presence of curcumin treatment. Unfortunately, neither the mRNA nor the protein level of LPR6 was changed . We observed similar pattern in these proteins in another colon cancer cell line HCT116 , which confirmed that miR-130a functioned to antagonize curcumin’s anti-tumor effect.These data suggested that miR-130 might act to antagonize curcumin’s anti-tumor effect. We therefore examined the serum level of miR-130a in mice treated with curcumin. We divided the mice into two sub-groups: short-lived group  and long-lived group . The serum level of miR-130a was significantly higher in the short-lived group than that of the long-lived group of mice (Curcumin, the major curcuminoid and the most active component in turmeric, is a biologically active phytochemical . Curcumiin vivo using a mouse model. Curcumin exerts its anti-tumor activity by inhibiting proliferation rather than promoting apoptosis of the colon cancer cells. Then we attempted to identify the signaling pathway involved in this process. In particular, curcumin affects several signaling pathways known to be important for solid and blood cancers, such as the PI3k/Akt-1/mTOR pathway (In this study, we first confirmed the anti-tumor activity of curcumin in colon cancer  pathway , the Ras pathway , the GSK pathway , p53 act pathway , and NF- pathway . In addi pathway . TherefoNext, we investigated the mechanism underlying curcumin effect on Wnt signaling. In addition to the gene expression studies , we notiSubsequently, we found that miR-130a could repress the expression level of Nkd2, which could be up-regulated by curcumin. These results suggested that miR-130a may regulate the Wnt pathway by inhibiting Nkd2. However, none of the predicted target genes of miR-130a were found to be potential indirect regulators of Nkd2, therefore the exact mechanism under this inhibition still elusive, and further investigations are warranted. It has been reported that miR-130a expression is disregulated in several types of cancer , includiFurthermore, we have verified that curcumin exhibits anti-tumor activity in colon cancer, which was exerted by inhibiting the Wnt/β-catenin signaling pathway to suppress cell proliferation in the colon cancer cells. Our results further suggested that curcumin could affect levels of several miRNAs in colon cancer cells. Among these miRNAs, miR-130a may be important for cancer formation because overexpressing miR-130a could restore the proliferation of curcumin-suppressed cancer cells back to normal. Meanwhile, the expression of Wnt signaling pathway component, β-catenin, was also restored to similar level as the original cancer cells. These results indicated that curcumin inhibited the Wnt signaling by repressing the expression level of the miR-130a. Furthermore, we tested the serum level of miR-130a in the experimental mice. We found that the short-lived mice had higher level of miR-130a in their serum than the long-lived mice. This suggested that miR-130a might contribute to the resistance of colon cancer cells to chemotherapy. Our study supports miR-130a as a novel target in the treatment of colon cancer. Taken together our earlier clinical study where high miR-130a expression level was significantly associated with poor clinical outcome , resultsCurcumin suppresses colon cancer by inhibiting Wnt/β-catenin pathway via miR-130a. MiR-130a may serve as a new target for CRC treatment.HD, RS, JT, LH, HS, HC, and YW performed the experiments and analyzed the data. TW wrote the manuscript and designed the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In situ hybridization histochemistry showed markedly elevated IGFBP-3 expression in the MPOA and the arcuate nucleus in rat dams. Prolonged intracerebroventricular injection of IGF-I or antagonism of brain IGFBP-3 with an inhibitor (NBI-31772) using osmotic minipumps increased pup retrieval time, suggesting reduced maternal motivation. Suckling-induced prolactin release and pup weight gain were also suppressed by IGF-I, suggesting reduced lactation. In addition, IGF-I-induced tyrosine hydroxylase expression and its specific phosphorylation in tuberoinfundibular dopaminergic neurons suppress prolactin secretion. Thus, IGF-I may inhibit both behavioural and lactational alterations in mothers. Neurons in the MPOA and arcuate nuclei express IGFBP-3 during the postpartum period to neutralize IGF-I effects. IGFBP-3 can prevent the blockade of maternal behaviour and lactation exerted by IGF-I, suggesting a novel modulatory mechanism underlying the behavioural and hormonal effects during central maternal adaptations.Adaptation to motherhood includes maternal behaviour and lactation during the postpartum period. The major organizing centres of maternal behaviour and lactation are located in the hypothalamic medial preoptic area (MPOA) and the arcuate nucleus, respectively. Insulin-like growth factor I (IGF-I) is an effector of the growth hormone axis; however, its function in the brain is largely unexplored. We identified increased maternal IGF binding protein-3 (IGFBP-3) expression in preoptic rat microarray data and confirmed it by RT-PCR.  Prolactin secretion is controlled by the inhibitory effect of dopamine produced by the tuberoinfundibular dopamine (TIDA) neurons. These neurons are located in the arcuate nucleus and project to the external zone of the median eminence and excrete dopamine into the pituitary portal blood vessels2. The modulators of prolactin release are known to affect the TIDA neurons; however, the major intrinsic regulators of the switch in the mode of these neurons, which permits the dramatically increased prolactin secretion required for lactation, need to be elucidated3.Postpartum physiological and behavioural changes are important parts of mammalian reproduction, and they can be investigated using the rat as an animal model. Non-maternal females do not care about pups or even attack them, while mothers demonstrate well-defined maternal behaviours, e.g., nest building, pup retrieval to the nest, nursing, and decreased anxiety, in addition to lactation. These marked changes are the consequences of maternal adaptation of the central nervous system. Lactation is known to be driven by prolactin secreted from the pituitary5 but are not required for it7. Rather, maternal behaviour is controlled by a complex neuronal network9 in which the medial preoptic area (MPOA) plays a central role10. The density of active neurons is dramatically induced in the MPOA of parenting females9. Furthermore, lesions of the MPOA abolish the nest building and retrieving components of maternal behaviour in lactating females13, while electrical and optogenetic stimulation of this area enhances maternal responsiveness14. Although the molecular mechanisms of maternal motivation are unknown, gene expression alterations have been hypothesized to be involved15. Previously, we carried out a microarray study of the preoptic area and identified amylin as a maternally induced neuropeptide16. We validated and functionally characterized amylin in mother rats17 but did not evaluate other genes with altered mRNA expression. In the present study, we identified significantly altered genes in our previous microarray study16, compared them with previous microarray data18, and investigated the maternal function of a gene altered in both studies, insulin-like growth factor binding protein-3 (IGFBP-3).Prolactin and other hormones contribute to the initiation of maternal behaviours19. By binding IGF with high affinity in a functionally inactive complex, IGFBP-3 can inhibit the effects of IGF-I. IGFBP-3 overexpressing mice show intrauterine and postnatal growth retardation, confirming the IGF-neutralizing role of IGFBP-320. In addition, IGFBP-3 may also have IGF-independent effects23. IGFBP-3 is normally expressed in the adult central nervous system (CNS) at a low level, mainly in non-neuronal cells, and the effects of IGFBP-3 on the CNS are largely unknown28.Insulin-like growth factor binding protein-3 (IGFBP-3) binds insulin-like growth factor-I (IGF-I) in the plasma and extracellular space. Although 6 other IGFBPs exist, IGFBP-3 is the major carrier of IGF and binds the majority of IGF-I in circulation29. IGF-1 signalling in the brain supports neuronal survival and neuroprotection during the development of the CNS and in adults following brain injuries30. In the hypothalamus, IGF-I can feedback to the growth hormone axis but may also affect reproductive neuroendocrine functions33. However, the effects of IGF-I on lactation and prolactin release, an important aspect of reproduction, have not been investigated.IGF-I is mainly produced by the liver and is related to the growth hormone axis. IGF-I is released into the circulation and can reach the central nervous system via the blood-brain-barrier and blood-cerebrospinal fluid barrier. In addition, IGF-I is also expressed in the brain. The actions of IGF-1 are mediated by a cell surface receptor, type 1 IGF receptor (IGF-1R), which is the major transducer of IGF signalsin vivo and in vitro.In this study, we first established that the expression of IGFBP-3 was significantly elevated in lactating rat mothers compared with that in mothers whose pups were taken away right after parturition (pup-deprived mothers). Second, we revealed the involvement of IGFBP-3 in the control of maternal behaviour with an IGFBP-3 antagonist. We also showed that the effect of IGF-I treatment was similar that of the IGFBP-3 antagonist. Third, we described the expression pattern of IGFBP-3 in the hypothalamus and identified two regions, the medial preoptic area and the arcuate nucleus, where its expression was markedly elevated in rat dams. Then, we demonstrated that the IGF-IGFBP-3 system regulates suckling-induced prolactin release. Furthermore, we identified the underlying mechanisms of the effects of IGF-I on prolactin-regulating tuberoinfundibular dopaminergic neurons both 16 and compared the results of this analysis with a recent microarray study18. We identified 21 genes with highly significant differences in expression levels between the maternal and non-maternal groups (p < 0.007) in our previous experiment  were 2–4 times higher in rat dams than those in the pup-deprived control group. Of these genes, insulin-like growth factor binding protein-3 (IGFBP-3) was also up-regulated in the study of Driessen et al. 2014. Therefore, we choose to further investigate the maternal function of this protein.To determine the genes that show altered expression in mothers, we re-evaluated our previous microarray study in the preoptic area of mother ratsin situ hybridization histochemistry to determine its distribution in the preoptic area  received ACSF. Then, NBI-31772 dissolved in 1% dimethyl sulfoxide (DMSO) in ACSF was administered i.c.v. to another group. Their control group received 1% DMSO in ACSF i.c.v. We performed a pup-retrieval test on the 6th day postpartum and found a markedly elevated pup retrieval time in the IGF-I- and NBI-31772-treated groups of mothers  for 5 days, 3 × 1 hour/day, 20 observations/hour and did not find any significant difference between the groups  via osmotic minipumps implanted in rat mothers on the 2ers Fig. . These mIn situ hybridization histochemistry revealed that the expression of IGFBP-3 is not confined to the preoptic area within the hypothalamus; the dorsomedial subdivision of the arcuate nucleus and the paraventricular hypothalamic nucleus (PVN) also contained IGFBP-3 mRNA  is the main immunohistochemical marker of dopaminergic neurons in the arcuate nucleus, which regulates the secretion of prolactin from the pituitary. Therefore, we combined IGFBP-3 eus Fig. . We founth postpartum day. Before the pups were removed for 4 h, the prolactin concentrations were 166.3 ± 30.3 and 103.96 ± 22.35 ng/ml in the ACSF- and IGF-I-treated groups, respectively. The plasma prolactin concentration was reduced to basal levels by the end of a subsequent 4-hour separation period , which play a crucial role in the regulation of pituitary prolactin secretion, and in dopaminergic cells in the zona incerta (A13 cell group), which do not participate in the control of prolactin secretion. TH expression was quantified using densitometry in the brains of IGF-I- and ACSF-treated rat dams. A twofold elevation in TH mRNA expression was found in IGF-I-treated mothers in the arcuate nucleus compared with that in the control group  or control medium. IGF-I administration significantly (p = 0.048) enhanced the TH mRNA expression – measured by real time RT-PCR – compared with that in the control cultures, which were treated with control medium Fig. .Figure 7th day, half of them were incubated in IGF-I-containing medium for 30 min at the same concentration as described above. Using Western blotting with phosphorylation-site specific antibodies, we determined that IGF-I stimulated a significant increase in the phosphorylation of the Ser 31 residue when compared with that in cells incubated under basal conditions. In contrast, IGF-I did not enhance the phosphorylation of the Ser 40 residue , which was elevated in mother rats in both studies. Because of the high rate of false positive data in microarray experiments34, we measured the mRNA level of IGFBP-3 in rat dams and pup-deprived controls with 2 independent methods, RT-PCR and in situ hybridization histochemistry, as IGFBP-3 induction has not been validated in the previous studies. Both methods validated the increased IGFBP-3 expression level in the preoptic area in lactating rat dams. The confirmed maternal elevation in IGFBP-3 expression and its distribution in the region of the preoptic area involved in maternal behaviour, the medial preoptic area35, suggest its involvement in the control of maternal behaviour. There are several different neuronal populations in this area that contribute to this regulation network, including galanin-, amylin- or melanin-concentrating hormone (MCH)-expressing neurons36. Of these populations, the distribution of IGFBP-3 in the MPOA is similar to the expression pattern of MCH. The expression of MCH increases gradually until the weaning of pups37 and has been suggested to be involved in the termination of maternal responsiveness during the late postpartum period38.We have now evaluated our previous microarray study, which was focused on a single genein situ hybridization histochemistry, suggesting additional functions. The role of IGFBP-3 in the PVN is unlikely to be related to maternal modifications as the expression level did not change in mothers. This finding also suggests that the induction of IGFBP-3 may be specific to certain cell types. Indeed, IGFBP-3 was induced in the dorsomedial subdivision of the arcuate nucleus, specifically in TH-positive dopaminergic cells. These cells are known to regulate prolactin release from the pituitary39, which suggests an additional role for IGFBP-3 in lactation. The IGFBP-3 distribution data in the hypothalamus have not been reported before. A previous study focusing on the effect of hypoxia on IGFBP-3 reported its expression in the brain but described only cortical and thalamic locations40.Apart from the preoptic area, IGFBP-3 expression was also detected in other hypothalamic sites by 41. These infusions presumably increased the hypothalamic bioavailability of IGF-I. Although peripherally administered IGF-I reduces the brain level of IGF-I42, we used continuous central infusions to ensure its permanently high bioavailability in the hypothalamus. To administer the drugs, we used osmotic minipumps with a continuous 0.5 μl/hour flow rate for a maximum of 14 days. Another reason why we preferred prolonged IGFBP-3 inhibition is that slow, neuroplastic effects can also be detected. For example, acute IGF-I and NBI-31772 administration have anxiolytic and antidepressant-like effects43, while their prolonged administration can affect neuronal plasticity and neural survival in dopaminergic neurons45. We did not detect the anxiolytic effects of these agents following their long-term presence as neither affected the number of open arm entries in the elevated plus-maze test, a marker of anxiety-like behaviour.The distribution of IGFBP-3 suggests that it may participate in the control of maternal behaviours and lactation. IGFBP-3 binds IGF-I with high affinity, thereby reducing the level of bioavailable IGF-I. To examine the role of IGFBP-3 in the regulation of maternal behaviours and lactation, we inhibited this IGF-sequestering effect with continuous and prolonged i.c.v. infusion of either IGF-I or an IGFBP ligand-binding inhibitor, NBI-3177235, our results suggest that elevated expression of IGFBP-3 in the MPOA in lactating dams contributes to maternal motivation. Different factors are known to contribute to the initiation and maintenance of maternal behaviour. While peripartum hormonal changes initiate and potentiate the onset of maternal motivation, they are not required for maintenance of maternal care. Continuous exposure to pups and afferent stimuli from them are sufficient for supporting the maintenance of maternal motivation during the postpartum period15. The neurochemical regulatory mechanisms, which contribute to ongoing maternal care, are much less established. Based on our results, IGF-I and IGFBP-3 appear to contribute to the maintenance of maternal behaviours, thereby suggesting a previously unknown mechanism involved in maternal regulations. The lack of effects of prolonged IGF-I and NBI-31772 on activity in the elevated plus-maze test indicates that reduced maternal motivation was not a consequence of altered state of anxiety, which itself could affect maternal behaviour.The effect of IGFBP-3 inhibition in the brain on maternal behaviour was examined. The mothers showed several aspects of normal behaviour, demonstrating that they can perform maternal behaviours. However, we measured an increased latency to carry the first pup and the last pup into the nest following the sequestration of IGFBP-3. Since moving the pups into the nest is the most frequently used indicator of maternal motivation as a goal-directed behaviour46. We observed a high degree of elevation in control mothers, which received ACSF injection. However, prolactin levels increased to a significantly lesser degree after the sequestration of IGFBP-3, supporting a role of IGFBP-3 in the regulation of prolactin secretion. Since prolactin increased significantly even in the presence of IGF-I in response to suckling, it is not possible to determine if a higher concentration of IGF-I could have blocked it completely or if prolactin has an IGF-I-independent component. At the same time, as prolactin levels were reduced in the presence of IGF-I, the body weight of pups gained during 1 hour of suckling was also decreased in the IGF-I group compared with that in the controls. These are new findings as hypothalamic IGF-I and IGFBP-3 have not been shown to control prolactin secretion in mothers. Previous studies have revealed that IGF-I acts on lactation at the level of the pituitary by supporting the differentiation and proliferation of lactotroph cells48. The involvement of hypothalamic IGF-I in reproductive neuroendocrine functions has been suggested before; it plays a role in the regulation of gonadotropin-releasing hormone (GnRH) cell function. Infusion of IGF-1 stimulated secretion of GnRH49, while its antagonism impaired oestradiol-positive feedback and luteinizing hormone (LH) surges33.Based on the induction of IGFBP-3 in TH neurons of the arcuate nucleus, we also addressed its role in lactation. In rodent mothers, lactation is driven by the suckling pup stimuli, which evokes a dramatic increase in serum prolactin levels52. IGF-I gene therapy in female rats reversed their hypothalamic DA dysfunction and hyperprolactinemia53. In line with these data, IGF-I enhanced TH expression in our hypothalamic cell culture experiments and in lactating rat dams. In dopaminergic neurons, activated ERK1/2 proteins enhance the transcriptional activation of TH by nuclear receptor related-1 (Nurr1)55, which therefore represents a possible mechanism of the effect of IGF-I on TIDA neurons in our study. In addition, we also found an increase in the level of phosphorylation of the TH enzyme at the Ser 31 phosphorylation site by IGF-I in vitro. This finding suggests rapid enhancement of dopamine synthesis by IGF-I as phosphorylation of TH results in an increased catecholamine production56. This rapid action of IGF-I on dopaminergic neurons has not been previously described. Rat TH can be phosphorylated at the four following different sites: Ser 8, 19, 31, 40, among which IGF-I action is selective for site 31 over 40. ERK1/2 serine kinases are mainly responsible for Ser 31 phosphorylation57, suggesting that IGF-I may have acted via ERK1/2 serine kinases to induce phosphorylation of TH.Binding of IGF-I to its receptor results in autophosphorylation of the receptor, which in turn initiates a cascade of cellular signal transduction events. One key step is the binding of insulin receptor substrate (IRS)-1 to phosphotyrosine residues on the receptor. IRS-1 then acts as a docking protein for the downstream signal transduction components, including the Ras/ERK1/2 and PI3K/Akt pathways. IGF-I supports the survival of dopaminergic neurons through these two pathways, which has mostly been investigated in the nigrostriatal dopamine system58. IGF-I in this region could originate in neurons, which express IGF-I in the hypothalamus59, and in the circulation as IGF-I can reach the hypothalamus by crossing the blood-brain-barrier or blood-cerebrospinal fluid barrier60. IGF-I in the plasma shows decreased levels during pregnancy because of reduced growth hormone (GH) receptor expression in the liver. After delivery, circulating IGF-I reaches its original, higher levels61. The suckling stimulus is known to cause a rapid and transient increase in plasma GH62 but does not result in any significant difference between lactating and pup-deprived animals61. These data suggest that the IGF-I concentration in the hypothalamus does not differ between lactating, pup-deprived mothers and virgin females, so its effects must be modified by other mechanisms that alter its concentration.To exert these actions, IGF-I is present in the hypothalamic extracellular space at a relatively high concentration63. We propose a similar mechanism in mother rats based on the existence of intrinsic IGF-I and IGFBP-3, which compose a system to regulate maternal changes in the brain  is also known66. In the hypothalamus, SHBG expression shows alterations according to the reproductive state, so it may play role in modulating the central actions of ovarian steroids67. However, we also established state-dependent induction of IGFBP-3 to regulate the actions of IGF-I actions on these steroids. This hypothesis represents a novel mechanism in neuroendocrinology; neurons can block the effects of a hormone by inducing the expression of its binding protein. Nevertheless, the recently suggested role of IGF-independent effects of IGFBP-323 in maternal brain alterations cannot be excluded.The regulation of the actions of a hypothalamic hormone by its binding proteins has also been previously suggested in other systems. For example, the corticosteroid-binding globulin expressed in the brain can direct the action of the stress hormoneIn conclusion, the major finding of this study is that prolonged IGF-I treatment reduced suckling-induced prolactin release and maternal responsiveness. In turn, IGFBP-3 expression shows marked elevation in well-defined regions of the maternal brain, including the MPOA and the arcuate nucleus. The role of this elevated IGFBP-3 may be the neutralization of the effect of IGF-I on distinct neurons by binding and sequestering it. Preventing the binding of IGFBP-3 to IGF-I with the ligand inhibitor NBI-31772 significantly suppressed maternal motivation. In addition, IGF-I also inhibited suckling-induced prolactin release. IGF-I can exert this action by increasing the dopamine release of TIDA neurons as IGF-I stimulated TH expression and phosphorylation. IGFBP-3 can reduce this action of IGF-I as TIDA neurons in the arcuate nucleus expressed a markedly elevated level of IGFBP-3 in lactating mothers. Thus, IGFBP-3 functions as an intrinsic regulator of lactation by switching TIDA neurons to a mode that permits induction of PRL release from the pituitary. Therefore, the IGF-I - IGFBP-3 systems can play an important role in the different aspects of the adaptation of the female brain to motherhood by a novel neuroendocrine mechanism.All animal experimentations were approved by the Animal Examination Ethical Council of the Animal Protection Advisory Board at the Semmelweis University, Budapest, and met the guidelines of the Animal Hygiene and Food Control Department, Ministry of Agriculture, Hungary. A total of 85 female Wistar rats  were used in this study . Then, cDNA was synthesized using SupersciptII (Invitrogen) as suggested in the kit protocol. The cDNA was subsequently diluted (10x), and 2.5 µL of the resulting cDNA was used as template in PCR reactions using SYBR Green dye . The PCR reactions were performed with iTaq DNA polymerase  and GAPDH was used as housekeeping gene. The primers were: ACAGCCAGCGCTACAAAGTT and GCGGTATCTACTGGCTCTGC for IGFBP-3, and GCTACCGAGAGGACAGCATC and GCACCATAAGCCTTCAGCTC for TH, and TGCCACTCAGAAGACTGTGG and GTCCTCAGTGTAGCCCAGGA for GAPDH. Cycle threshold (Ct) values were obtained from the linear region of baseline adjusted amplification curves. The GAPDH related values were calculated using the following formula: log(Ct(GAPDH) − Ct(IGFBP3 or TH)). Statistical analyses were performed by unpaired t-test for comparisons of the two different groups.Real-time RT-PCR was carried out as described previouslyin situ hybridization probes was performed as described previously68. Briefly, PCR products of amplification of IGFBP-3 and TH were produced from hypothalamic cDNA using the following primer pairs for IGFBP-3: A: ACAGCCAGCGCTACAAAGTT and GCGGTATCTACTGGCTCTGC, B: CCTTGTTGGAGACCCTGGTA and TCACACCCTGTATTGCCAGA; and the following for TH: GCTACCGAGAGGACAGCATC and GCACCATAAGCCTTCAGCTC. The PCR products were purified from gel, inserted into TOPO TA cloning vectors (Life Technologies) and transformed chemically into competent bacteria. Selected plasmids were applied as templates in PCR reactions, using the primer pairs specific for IGFBP-3 and TH, respectively, with the reverse primers also containing a T7 RNA polymerase recognition site. At the end, the identities of the cDNA probes were verified by sequencing.Preparation of the In situ hybridization histochemistry was processed as described previously16. Briefly, serial coronal sections (12 µm) were cut using a cryostat from bregma level + 3.5 mm to −6 mm, mounted on positively charged slides , dried, and stored at −80 °C until use. Antisense [35 S]UTP-labelled riboprobes were generated using T7 RNA polymerase of the MAXIscript In vitro transcription kit  from PCR-amplified fragments of the cDNA subcloned into TOPO TA vectors. Tissue was prepared using an mRNA-locator Kit (Ambion) according to manufacturer’s instructions. For hybridization, we used 80 µl hybridization buffer and 1 million DPM of labelled probe per slide. Washing procedures included a 30 min incubation in RNase A, followed by decreasing concentrations of sodium-citrate buffer (pH = 7.4) at room temperature, and then at 65 °C. After drying, slides were dipped in NTB nuclear track emulsion , stored for 3 weeks at 4 °C for autoradiography. Then, the slides were developed and fixed with Kodak Dektol developer and Kodak fixer, respectively, counterstained with Giemsa, dehydrated, and coverslipped.To describe the expression pattern of IGFBP-3 and also to measure the expression level of IGFBP-3, brains of 6 primiparous lactating mothers and 6 age-matched pup-deprived mothers were removed at 11 days postpartum. In addition, brains of 4 IGF-I and 4 ACSF-treated rat mothers were removed at the end of the treatment, at 14 days postpartum to examine the effects of prolonged i.c.v. IGF-I administration on TH expression levels. The fresh tissue was quickly frozen on dry ice. Dark-field photomicrographs were taken of the sections where IGFBP-3 and TH signal was the highest in the medial preoptic area, and arcuate nucleus and zona incerta, respectively, using a 10x objective. Each image was divided into 2 halves with identical size, such that one half contained the observed autoradiography signals, while the other half served as background control. The pixel number of these areas was calculated using ImageJ 1.47 v  software. The difference between the 2 values  was used to quantify IGFBP-3 and TH mRNA levels. mRNA levels in the 2 groups were compared using two-tailed unpaired t-test.nd day postpartum, rats were divided into 4 groups to determine the role of IGF-I system in maternal behaviour and TH expression level: icv. infusion of ACSF (n = 10), IGF-I dissolved in ACSF (n = 10), 1% DMSO in ACSF (n = 8), NBI-31772 (n = 8) dissolved in ACSF containing 1% Dimethyl sulfoxide (DMSO). In another experiment, 7 IGF-I-treated vs. 8 ACSF-treated (control) mothers were used to determine the role of IGF-I system in prolactin release. Osmotic minipumps injecting continuously for 14 days  were used loaded with IGF-I , NBI-31772 , ACSF , or 1%DMSO in ACSF connected to cannulae according to manufacturer’s guide. For intracerebroventricular (i.c.v.) implantation of the cannulae, rats were anesthetized with 0.2 ml xylazine and 0.3 ml ketamine and fixed in a stereotaxic apparatus. The skin was cut over the skull and a hole of about 1 mm diameter was drilled into the left side of the skull above the lateral ventricle positioned at the following coordinates: antero-posterior, −0.5; lateral, 1.4; ventral, 3.6 mm. Cannulae  were inserted into the lateral ventricle and fixed to the skull with cranioplastic cement and the pumps placed subcutaneously (s.c.) at the back of the animal. After the operation, Tardomyocel® comp. III antibiotics (0.1 ml/kg body weight) was given s.c. to the animals for 5 days to prevent infections.On the 269. Briefly, the undisturbed maternal behaviour was observed for three 60 min daily observation periods, 5 days long, starting on 4th day postpartum, after one day recovery following the implantation of osmotic minipumps. Observations were performed at two periods during the light phase  and at one period during the dark phase . Within each observation period, the behaviour of each mother was scored 20 times spaced 3 min each one (20 observations × 3 periods per day × 5 days = 300 observations/mother) as present or absent. The following behaviours were scored as present or absent: high kyphosis (HK): mother nursing pups in an arched-back posture with rigid limbs; licking/grooming (LG): mother licking–grooming any pup ; prone nursing (PN): mother nursing in a “blanket” posture in which the mother just lies over the pups, but did not have her back arched and there was no obvious extension of her legs; supine nursing (SN): mother nursing in a “passive” posture in which the mother lies on her back or side while the pups are nursed; out of the nest (0): mother out of the nest . Number of each behaviour/300 observation ratio was calculated for every mother. Normality was tested with Kolmogorov-Smirnov test while difference of the mean was analyzed with unpaired two-tailed t-test.We used 36 mother rats injected i.c.v. with osmotic minipumps to investigate their undisturbed maternal behaviour. The behaviour of each dam was recorded as described previouslyWe used 36 mother rats injected i.c.v. with osmotic minipumps for testing. On postnatal day 6, all pups were separated from their mothers for 10 min. Subsequently, 3 pups were returned to the mother’s cage in 3 different corners of the cage away from the nest. The mother was visually observed for 5 min. The time required for the mother to carry the first and the third pup to the nest was recorded. Normality was tested with Kolmogorov-Smirnov test. Results were analyzed with two-tailed Mann-Whitney-, or unpaired t-test with Welch correction.To measure anxiety, dams were exposed to EPM on postnatal day 7 during the afternoon. The EPM was made of plastic, painted black and elevated 80 cm above the floor . Surface of maze was washed with water, alcohol and dried before the next animal was introduced. The duration of the test was 5 min. Rats were introduced in the centre of the maze facing a closed arm. Percentage of time spent in open arms and percentage of open arm entries (number of open arm entries/number of open plus closed arm entries) were calculated and used as measures of anxiety (entry: at least two third of the body in an arm). Normality was tested with Kolmogorov-Smirnov test while difference between the means was analyzed with two-tailed unpaired t-test.In situ hybridization histochemistry was performed as described above except that 20 mm thick sections were used. Following hybridization with IGFBP-3 probe and before dipping the sections in nuclear track emulsion, TH immunohistochemistry was carried out in humid chambers. Slides were first treated with mouse anti-TH antibody , then in horse anti-mouse IgG  for 1 h, then in ABC complex  for 2 h. Finally, the sections were visualized with 3,3-diaminobenzidine (DAB) reaction.Brains of 5 lactating mother rats perfusion fixed on postpartum day 11 were used to examine the expression of IGFBP-3 in dopaminergic neurons. Sections were examined using an Olympus BX60 light microscope equipped with a dark-field condenser. Images were captured at 2048 × 2048 pixel resolution with a SPOT Xplorer digital CCD camera  using 4–40 X objectives. Images were adjusted using the “levels” and “sharpness” commands in Adobe Photoshop CS 8.0. Full resolution of the images was maintained until the final versions, which were adjusted to a resolution of 300 dpi.th postpartum day - under ketamine-xylazine anaesthesia (described above) – 15 dams  received 25-mm-long sterile polyethylene jugular cannulae (Plastics One). A ventral cervical skin incision was made right of the midline with its caudal terminus at the level of the clavicle. The right common jugular vein was mobilized, and cannulae were inserted into the vessel and secured in place with suture. Incisions were made on the skin at the midline between the scapulae, and jugular cannulae were pulled through the scapular incisions. The cannulae were filled with heparinized saline and sealed with metal pins.On the 13On the next day we obtained blood via jugular cannulae to measure serum prolactin levels induced by suckling: first before taking away the pups for 4 h, second before returning the pups (baseline control) and also 5, 15, 30, 60 min after the return. The volume of blood taken was 0.3–0.3 ml each time, and the same amount of heparinized saline was injected back into the circulation. Blood was centrifuged at 4 °C for 10 min at 12.000 g and the supernatant was stored at −20 °C for measurement of prolactin plasma concentrations by radioimmunoassay.70. Briefly, the chloramine-T method was used for iodination, and protein A  was used to separate bound and free hormone. LKB Clinigamma software was used for data collection and calculations for curve fitting. Within-assay variance was 10%. Between-assay variance was 14%. The sensitivity of the prolactin assay was 0.5 ng/ml rat plasma (or 25 pg prolactin). All samples were analyzed in duplicate using 50 ml of plasma for each measurement.Prolactin assay was performed as described previouslyStatistical analyses were performed using Prism 5 for Windows . Normality was tested with Kolmogorov-Smirnov test. For the suckling experiment, plasma prolactin levels of the 2 groups were compared using repeated measures two-way ANOVA to evaluate whether suckling and prolonged administration of IGF-I had an effect on the prolactin level. For posthoc comparisons, Newman-Keuls test were used.5 cells/ml and plated out at 0.5 ml/well onto 8 wells of a polylysine coated 24-well culture plate, which were then transferred to a 5% CO2 humidified incubator at 37 °C. After the first 24 h in culture, half of the medium (250 µl/well) was removed and changed to 25 µg/ml IGF-I  in 4 wells and DMEM-F12-B27-P/S in other 4 wells, as controls. Half of the medium was replaced every day as described above and cells were used for examination on the 5th day.10 pathogen-free newborn Wistar rats (Charles-River Laboratories) were decapitated right after birth and the mediobasal hypothalamus was excised. The tissue blocks were immediately transferred to an ice cold medium containing 2% B27, 49% Dulbecco’s modified Eagle’s medium, 49% F12 medium and penicillin-streptomycin . First, tissue was homogenized and centrifuged for 4 min on 3000 rpm. Second, the supernatant was removed and 2 ml 0.1% Trypsin (diluted in DMEM) added to the pellet. Third, after 4 min incubation in a 37 °C water bath with Trypsin, 2 ml 0.5% DNase and 1% Bovine Serum Albumin (diluted in DMEM-P/S) was added to the digested tissue, mixed and centrifuged for 4 min at 3000 rpm. After this, supernatant was removed again, and 2 ml DMEM-F12-B27-P/S was put to the pellet, mixed and centrifuged for 4 min at 3000 rpm. This step was repeated twice and the resultant cell pellet was dispersed in DMEM-F12-B27-P/S at a density of approximately 3.9 × 10After removal of the medium, cells were washed twice with sterile PB solution and then mixed with RIPA  + Protease Inhibitor  using a pipette tip. Cells were collected in RIPA + and centrifuged on 4 °C at 16000 rpm. Protein concentration was measured and equilibrated to 1 µg/µL. After that Laemmli sample buffer  was added to the removed supernatant and heated at 90 °C for 5 min. After running the gels and transferring the proteins to membranes , the membranes were treated with 3% BSA solution for 2 h. Then, primary antibodies were added to membranes for 24 h  and secondary for 1 h . Membranes were treated with Clarity™ Western ECL Substrate (Bio-Rad) for chemiluminescence detection, which was performed with ChemiDoc™ MP Imaging System (Bio-Rad). Intensity of bands was calculated using ImageLab software (Bio-Rad). Ratio of phospho TH – TH intensity was calculated using the following formula: (phospho TH intensity/-tubulin-III intensity)/(TH intensity/β-tubulin-III intensity) and analysed with two-tailed unpaired t-test.


Brugia malayi causes the human tropical disease, lymphatic filariasis. Microfilariae (Mf) of this nematode live in the bloodstream and are ingested by a feeding mosquito vector. Interestingly, in a remarkable co-evolutionary adaptation, Mf appearance in the peripheral blood follows a circadian periodicity and reaches a peak when the mosquito is most likely to feed. For the remaining hours, the majority of Mf sequester in the lung capillaries. This circadian phenomenon has been widely reported and is likely to maximise parasite fitness and optimise transmission potential. However, the mechanism of Mf sequestration in the lungs remains largely unresolved. In this study, we demonstrate that B. malayi Mf can, directly adhere to vascular endothelial cells under static conditions and under flow conditions, they can bind at high (but not low) flow rates. High flow rates are more likely to be experienced diurnally. Furthermore, a non-periodic nematode adheres less efficiently to endothelial cells. Strikingly C3, the central component of complement, plays a crucial role in the adherence interaction. These novel results show that microfilariae have the ability to bind to endothelial cells, which may explain their sequestration in the lungs, and this binding is increased in the presence of inflammatory mediators. Brugia malayi and Wuchereria bancrofti. The circadian appearance of first-stage larvae or microfilariae (Mf) in the peripheral blood of their host is a phenomenon first observed by Patrick Manson in the late 19th century and has been well described. Indeed, the synchronisation of microfilarial appearance in the blood with the peak feeding time of the local mosquito vector population in different parts of the world, suggests a co-evolutionary adaptation for maximising parasite transmission [B. malayi Mf can bind to vascular endothelial cells (EC) in vitro and we suggest that active adherence of Mf to EC may be one mechanism by which Mf sequester in the lungs of the human host.Circadian rhythms are thought to maximise the fitness of parasites by increasing transmission, survival or reproductive capacity. Lymphatic filariasis is a major tropical disease, caused by the filarial nematodes, smission ,3. It issmission ,5,6. HowWuchereria bancrofti and Brugia malayi microfilariae, which are normally nocturnally periodic or sub-periodic [Edesonfilaria malayensis in the macaque, a rise in temperature rather than oxygen levels causes disappearance of the microfilariae from the periphery. (Even non-periodic filariae including Litomosoides carinii (now sigmodontis) respond to a temperature drop to accumulate in the peripheral blood, which is thought to enable uptake by their burrow-dwelling mite vectors). Administration of serotonin into several filarial-infected hosts also causes an immediate release of Mf from the lungs into the peripheral blood [B. malayi Mf secrete potent vasodilators such as, prostacyclin and prostaglandin E2 (PGE2), which may allow these parasites to modulate vasculature function in order to interact with the endothelial surface [In a number of papers in the 1960s, (reviewed in ) Hawkingperiodic , fall inperiodic . Conversal blood ,8. Inter surface ,11.Schistosoma mansoni eggs adhere to EC surface molecules, ICAM-1, VCAM-1 and E-selectin via egg surface-expressed Lewis X antigen-related carbohydrates [S. mansoni eggs, Plasmodium falciparum-infected erythrocytes also bind to ICAM-1, VCAM-1 and E-selectin expressed on EC [P. falciparum-infected erythrocytes ligate to the endothelial surface molecules, CD36, thrombospondin, CD31, P-selectin and complement receptor 1 (CR1), as well as, chondroitin sulphate A (CSA) and hyaluronic acid [A close association of Mf with host vascular endothelium is not without precedent as several blood-borne parasites have been shown to form an intimate host-parasite relationship with these cells. hydrates . In commed on EC ,14,15,16nic acid ,18,19,20B. malayi Mf and vascular EC in vitro. Notably, B. malayi Mf bind to the surface of human umbilical vein EC (HUVEC) and they do so in far greater numbers than Mf of the filarial nematode, Litomosoides sigmodontis. Interestingly Kwa et al. (1991) also noted that B. malayi microfilariae can be seen adhering to mouse endothelial cells cultured from infected athymic mice [B. malayi Mf adherence, and indeed, C3 was seen deposited on the surface of Mf. However, the complement receptor CR1, which binds C3b and inactivated C3b (iC3b), and CR4, which binds only iC3b, do not appear to play a role in this interaction. Several other treatments also promoted B. malayi Mf adherence to HUVEC, although to a lesser degree than C3; these included stimulation of HUVEC with IFN-γ (but not TNF-α) and treatment of HUVEC with fixatives. The presence of diethylcarbamazine (DEC), which is known to cause the immediate release of Mf from the lungs into the peripheral blood of hosts in vivo, did not alter Mf adherence to EC in vitro [in vitro EC culture system, high flow rates, similar to those found in the host during the day, enabled Mf binding, while low flow rates, which may be found at night, did not result in Mf adherence. This study provides further clues to the mechanistic explanation for the retention of Mf in host non-peripheral blood vessels during their circadian periodicity.In this study, we modelled the interaction between mic mice . We founin vitro ,22,23,24Ethical approval was obtained from the East London Local Research Ethics Committee to collect human umbilical cords from mothers at the Royal London Hospital and blood from healthy donors. All study participants provided written informed consent. Parasite maintenance in animals and infection of animals was in accordance with the Home Office project licence (PPL 70/7243), which was approved by the Home Office under the Animal Scientific Procedures Act (1986), UK. The project was approved by the local Ethical Review Committee at the Royal Veterinary College, London UK.HUVEC were isolated from human umbilical cords, obtained from the Royal London Hospital, using a modified method ,26. In aBlood was collected from young healthy donors into tubes without anticoagulant, at around midday. After 3 hours at room temperature, serum was harvested following centrifugation at 2,057xg for 20 min. In several experiments, serum was heat-inactivated at 56°C for one hour in a water bath.B. malayi and L. sigmodontis Mf were obtained by peritoneal lavage with RPMI-1640 from infected gerbils (Meriones unguiculatus). . Mf were isolated by centrifugation over lymphocyte separation medium  [B. malayi microfilariae were exsheathed by incubating them for one to three hours in 20 mM CaCl2 in Earls phosphate-free balanced salt solution. Approximately 70% of Mf lost their sheaths after one hour, whereas 97% had exsheathed by 3 hours and they appeared highly motile and viable. In some experiments, 250,000 Mf were injected i.v. into groups of five 6–8 week old male BALB/c mice . Mice were tail bled at intervals at 11a.m., and the Mf obtained in 50μl of blood were counted following red blood cell lysis. Mice were anaesthetised i.m. with ketamine-xylazine.ls, USA) . B. malaB. malayi Mf. In experiments using HEK 293T and EaHy926, DMEM replaced the use of Medium 199 while in THP-1 experiments RPMI-1640 was used in place of Medium 199. All cell monolayers were fully confluent at the time of Mf co-culture. THP-1 and HUVEC were seeded for 48 hours and HEK 293T for 24 hours prior to co-culture with Mf. THP-1 are a monocytic cell line, and they are differentiated into macrophages using PMA (Sigma-Aldrich Ltd). THP-1 were stimulated with PMA (50ng/ml) 48 hours prior to adding Mf to the culture. Following co-culture, non-adhering Mf were washed off with pre-warmed medium and adherent Mf were counted using phase contrast microscopy. Further experiments were performed using a VenaFlux semi-automated microfluidic system to study Mf adhesion under shear flow conditions that are more physiologically relevant to in vivo blood flow rates [2. The ability of Mf to adhere to HUVEC under flow conditions was visualised using the VenaFlux software.Mf and HUVEC were co-cultured throughout this study in complete Medium 199 and RPMI (1:1) plus serum and 1% glucose (Sigma-Aldrich Ltd). Unless otherwise indicated, 20% FBS was used. HUVEC were cultured in HUVEC medium for 60 hours and thereafter the medium was replaced with co-culture medium containing 125,000 ow rates . The micIn some experiments, HUVEC were co-cultured with Mf in medium supplemented with different concentrations and types of serum or other additional additives. These included heat-inactivated or non-heat inactivated human serum, C3-depleted human serum, 250μg/ml mannan, 2.5μM DEC or 1nM melatonin (Sigma-Aldrich Ltd. The melatonin concentration used in this experiment corresponded approximately to the concentration of melatonin observed in human plasma at early night . In somek), W6/32 , L243 (mouse anti-human HLA-DRα), 6.5B5 (mouse anti-human ICAM-1) and DA6.231 (mouse anti-human HLA-DRβ) were grown in vitro. Supernatant was harvested, and antibody was purified over protein G Sepharose. Working dilutions of antibody were determined by staining HUVEC pre-stimulated with 10ng/ml human IFN-γ  for 48 hours. HUVEC were incubated for 30 minutes at the optimal working dilution. Mouse anti-I-Ek  was used as an isotype control antibody for W6/32 and L243 clone antibodies, while mouse anti-IgG1 (TNP-specific)  was used as an isotype control for D6.231 and 6.5B5 clone antibodies. Mouse anti-human CR1 (3D9 clone) antibody has previously been used to block the ligation of C3b to CR1 [In several experiments blocking antibodies were used against HUVEC. The monoclonal antibody hybridoma cells, HB32 (mouse anti-I-EHUVEC were treated with 7.8μM sodium azide (Sigma-Aldrich Ltd.) for 2h at 37°C to kill them. Cells were then washed 5 times with pre-warmed PBS to remove all traces of sodium azide before co-culture with Mf. For fixation, HUVEC were treated with 4% pre-warmed formaldehyde or pre-warmed 2% formaldehyde plus 2.5% glutaraldehyde and 25mM L-lysine (Sigma-Aldrich Ltd.) in PBS for 10 minutes before washing with PBS prior to co-culture with Mf.B. malayi Mf that had bound to HUVEC were fixed in 4% paraformaldehyde for 10 minutes at room temperature. The samples were dehydrated in 50% ethanol for 5 minutes, followed by 100% ethanol for 2 x 5 minutes. Then samples were adhered onto an SEM stub  using carbon adhesive discs . Prior to viewing, the prepared sample was coated with 20nm gold in a Quorum Q150T sputter coater . Digital images of the samples were captured under an FEI Quanta 200F SEM . The structural features of B. malayi Mf were characterized according to a previous publication [lication .For flow cytometry, antibodies were either used at the optimal working dilution as outlined above or according to the manufacturer`s instructions (mouse anti-human CD11c (Serotec)). Goat anti-mouse FITC conjugated antibodies (Sigma-Aldrich Ltd.) were used as a secondary antibody with unconjugated primary antibodies. Data were acquired using a FACS Canto II (BD Oxfordshire UK) and analysed with FlowJo software .p value < 0.05 was considered to be statistically significant. Data are presented as mean ± standard deviation.Student’s t-test for paired data and ANOVA was used for statistical analyses. A B. malayi Mf were incubated with HUVEC in vitro, a proportion of Mf were seen to attach to the cell surface. Mf used one end, and sometimes both ends simultaneously, to interact with the cell monolayer , but not during slow flow conditions (0.1 dyne/cm2)  was performed to study the binding of e detail . SeveralB. malayi is a nocturnally periodic nematode, melatonin could act as a trigger for the release of Mf from the lung capillaries into the peripheral blood. To investigate whether melatonin negatively affects the level of Mf adherence to HUVEC, Mf and HUVEC were co-cultured in the presence of 1nM melatonin. This concentration of melatonin corresponds to the amount observed in humans in the early hours of the morning [in vitro in static culture , found that although in some hosts B. malayi Mf did not respond to temperature fluctuations [B. malayi Mf counts in the peripheral blood [in vitro, Mf and HUVEC were incubated together at either 36°C or 37°C, however, no significant differences in the number of adhering Mf were found between the two temperatures in static culture  or PFA-glutaraldehyde . As fixeTo further investigate the potential cell surface molecules to which Mf may adhere, HUVEC were stimulated with IFN-γ or TNF-α, both of which are known to up-regulate a broad range of EC surface molecules ,37,38,39B. malayi Mf did not alter the level of expression of any of these surface markers in the presence or absence of IFN-γ  . In contof IFN-γ . To inveof IFN-γ , or ICAMof IFN-γ .B. malayi Mf adhere to HUVEC in vitro, the actual number of Mf adhering was low when heat-inactivated FBS was used as the serum supplement in the medium . In endemic areas, such as the South Pacific islands, where the main vector is day-biting, W. bancrofti Mf are diurnally periodic [Circadian periodicity of Mf is a phenomenon that has been widely reported for more than a century. It is thought that Mf periodicity maximises parasite fitness by increasing the chance of transmission. In parts of the world where the predominant mosquito vector is active at night, Mf of periodic . For theperiodic ,6. The mB. malayi from TRS labs that was originally nocturnally sub-periodic . This strain was later also found to be sub-periodic in mice [B. malayi has been shown to retain periodicity in rhesus monkeys, cotton rats, mice, gerbils and cats [B. malayi Mf had significantly more Mf in the peripheral blood in mice under general anaesthesia than those that were not anesthetised.Most labs worldwide now use the strain of  in mice ,43. Therand cats ,46,47,48and cats ,44. The in vivo. In contrast, Mf of the rodent filarial nematode, L. sigmodontis, adhered to EC only in low numbers. Strikingly, this rodent nematode is non-periodic in vivo.Interestingly, we found that Mf adhere to EC in static conditions, and they can also adhere to EC under high, but not low, flow rate conditions. This interaction could provide a potential mechanism for sequestration of Mf in the lung capillaries B. malayi Mf did not adhere to an embryonic kidney cell line (HEK 293T), while there was some binding of Mf to the monocytic cell line. Interestingly, adherence of B. malayi Mf to EC appeared to be with either the anterior and/or the posterior end of the Mf while the monocytic cells were more often attached to any part of the Mf surface in a manner reminiscent of their role as immunoprotective cells. A previous observation of Mf sticking to EC with one end has been made in mouse EC isolated ex vivo from an infected athymic mouse suggesting that this relationship may also occur in vivo [Trichuris muris, B. malayi Mf do not possess grooves on their dorsal surface, and thus, do not appear to be adapted for migration with the full length of their body through a cellular layer [2) or forces applied manually using a mechanical pipette-boy. in vivo . Scannin in vivo ,32. Althar layer . The attTrypanosoma cruzi synthesises melatonin, which is important for its growth and transformation, and, host-derived melatonin increases parasitaemia and maturation of both P. falciparum and P. chabaudi [Caenorhabditis elegans, is known to respond to melatonin via specific receptors; melatonin regulates locomotion and other homeostatic functions such as body phenotype and egg-laying in this nematode [in vitro model, however, neither melatonin nor lowered temperature altered the adherence of Mf to EC. One indication from our study is that reduced blood flow in the lung during vasoconstriction may control the appearance of nocturnally sub-periodic B. malayi Mf in the peripheral blood, as Mf were only able to adhere to EC under high flow conditions while under low flow conditions they did not bind.It remains uncertain how the circadian appearance of Mf in the peripheral blood is co-ordinated. The presence of melatonin and a lowered body temperature of 36°C are both associated with nocturnal physiology and several parasites have been shown to produce, as well as respond to, melatonin. chabaudi ,51. Intenematode . In our B. malayi Mf in the peripheral blood. The mechanism of this Mf release is not understood, however DEC is known to rapidly inhibit EC prostanoid metabolism [2-mediated vasodilation may result in vasoconstriction, increased resistance and therefore decreased blood pressure may result in Mf release into the periphery [in vitro we have shown that B. malayi Mf are less able to bind EC under hypoxic conditions. Additionally, during general anesthesia, which in respect of lower temperature and oxygen tension is similar to sleep, we observed higher Mf release into the peripheral blood in vivo. Overall, Mf retention in the lungs is likely to depend on vascular tone, which can be altered by oxygen tension and temperature, both of which are altered during sleep.Administration of DEC to infected individuals causes an immediate appearance of tabolism . Inhibiteriphery . This iseriphery  and in vFixatives that cause the cross-linking of proteins and/ or polysaccharides led to enhanced Mf adherence to EC suggesting that Mf bind to one or more cell surface molecule(s), that may be both preserved and exposed during fixing . Mf presS. mansoni schistosomulae can adhere to bovine brain microvascular EC, and the number of schistosomulae attaching is reduced in the presence of cytochalasin D, an inhibitor of actin polymerization [B. malayi Mf. However, attempts in our laboratory to block cytoskeletal rearrangement of HUVEC using cytochalasin B did not inhibit Mf adherence to HUVEC.While fixation of HUVEC may cross-link or unmask Mf binding sites on the cell surface, IFN-γ also induces cytoskeletal rearrangements that could in turn reveal novel sites to which Mf adhere . Interesrization . This inB. malayi Mf in vitro, and, in a mouse model, this C3 deposition was primarily mediated by MBL-A [L. carinii (now sigmodontis) Mf, which showed little adherence to EC does not bind complement [B.malayi Mf, C3 deposition appears to be particularly abundant at the nematode tips, which may explain the fact that Mf mainly adhere to HUVEC with their ends. Furthermore, exsheathment of Mf reduced their ability to bind EC. The sheath is known to contain mostly protein and some 5% carbohydrate, the most abundant component of which is N-acetyl-glucosamine while exsheathed Mf do not bind lectin and do not have surface N-acetyl-glucosamine [in vitro culture system as there is an absence of specific antibody, complement deposition on the surface of Mf is likely to be primarily via the MBL pathway, which can bind N-acetyl-glucosamine. Therefore in the absence of the sheath there will be little complement deposition via MBL.The key observation of this study is that a major component of the early immune response, C3 and/or downstream components of the complement cascade, have a crucial role in adherence of Mf to live vascular EC. It is known that C3 can be deposited on the surface of live by MBL-A ,59,60. Ncosamine . In our B. malayi Mf adherence to HUVEC may occur via the iC3b:CR1 axis [Onchocerca volvulus Mf following incubation with human serum, and factor H deposition (which can lead to iC3b formation) has been seen on the surface of both O. volvulus and Loa loa Mf [B. malayi Mf [HUVEC possess complement receptors, CR1 and CR4, on their surface. THP-1 possesses CR1, CR3 and CR4 while HEK cells do not have CR. CR1 ligates C3b with high affinity and iC3b with lower affinity, while CR4 ligates only iC3b ,63,64. ECR1 axis . Indeed,a loa Mf ,69. Intealayi Mf .in vivo and the fluctuations in the ability of Mf to adhere to EC in vitro. In particular we show a central role for C3, a major component of the immune response, in this in vitro interaction. Thus our work may promote a greater understanding of the mechanism of this highly evolved adaptation for increasing effective transmission.In conclusion, we report novel insights into the direct interaction of filarial Mf with vascular endothelial cells. Mf are known to display a circadian periodicity in their appearance in the peripheral blood which coincides with the mosquito vector’s feeding habits. Our studies reveal some parallels between the role of oxygen tension and vascular tone S1 Supporting InformationB. malayi Mf were co-cultured with HUVEC in medium supplemented with 10% human serum in static culture conditions. After 24 hours the co-culture was examined under a phase contrast microscope (magnification: 10 x). The video shows side-to-side movement of Mf bound to HUVEC with one or both ends. Non-binding Mf are also present.(MOV)Click here for additional data file.S2 Supporting InformationB. malayi Mf were co-cultured with HUVEC in medium supplemented with 10% human serum at a flow rate of 0.1 dyne/cm2. Non-binding Mf are observed flowing past the HUVEC monolayer.(MOV)Click here for additional data file.S3 Supporting InformationB. malayi Mf were co-cultured with HUVEC in medium supplemented with 10% human serum at a flow rate 1.0 dyne/cm2. They adhere with one or both ends and can be seen moving from side to side while bound to HUVEC. Non-binding Mf are observed flowing past the HUVEC monolayer.(MOV)Click here for additional data file.S4 Supporting Information2. Non-binding Mf are observed flowing past the HUVEC monolayer.Mf were co-cultured with HUVEC in medium supplemented with 10% human serum at a flow rate 1.0 dyne/cm(MOV)Click here for additional data file.S5 Supporting InformationB. malayi Mf were co-cultured with PMA-stimulated THP-1 in medium supplemented with 10% human serum. After 24 hours the co-culture was examined under phase contrast microscopy (magnification: 10 x). The video shows side-to-side movement of Mf that are adhering to the THP-1 monolayer while other THP-1 cells are seen adhering to the entire Mf surface.(MOV)Click here for additional data file.S6 Supporting InformationL. sigmodontis Mf were co-cultured with HUVEC in medium supplemented with 10% human serum. After 24 hours the co-culture was examined under phase contrast microscopy (magnification: 10 x). The video shows side-to-side movement of some Mf bound to HUVEC with one end.(MOV)Click here for additional data file.S7 Supporting InformationB. malayi Mf were co-cultured with HUVEC in medium supplemented with 10% human or foetal bovine serum. After 24 hours the Mf adhering to the cell monolayer were counted. Data are shown as the mean ± standard deviation of three independent experiments.(PPT)Click here for additional data file.


This study aimed to synthesize a novel microbial carrier to improve its adsorptive capacity for MTBE and biofilm formation, compared to the traditional granular activated carbon (GAC). A polypyrrole (PPy)-modified GAC composite (PPy/GAC) was synthesized, and characterized by Fourier transform infrared spectroscopy (FT-IR) and Brunauer-Emmett-Teller (BET) surface area analysis. The adsorption behaviors of MTBE were well described by the pseudo-second-order and Langmuir isotherm models. Furthermore, three biofilm reactors were established with PPy/GAC, PPy, and GAC as the carriers, respectively, and the degradation of MTBE under continuous flow was investigated. Compared to the biofilm reactors with PPy or GAC (which both broke after a period of operation), the PPy/GAC biofilm column produced stable effluents under variable treatment conditions with a long-term effluent MTBE concentration <20 μg/L. Pseudomonas aeruginosa and Acinetobacter pittii may be the predominant bacteria responsible for MTBE degradation in these biofilm reactors.Methyl  The widespread use of MTBE has resulted in increased reports of groundwater contamination, thus, the development of technology to eliminate MTBE contamination has become a priority [Methyl priority ,2. Adsorpriority ,4, howevpriority . MTBE capriority ,7,8,9; hThe biological activated carbon (BAC) process removes organic pollutants by a dual-removal mechanism: adsorption on GAC and biological degradation of the adsorbed and dissolved organic pollutants . The dua−, ClO4−, NO3−, and Br−, it can exhibit an anion-exchange capacity because of the high mobility of these ions in a polymer matrix. Specifically, when FeCl3 is used as the oxidant, its Cl− ions can act as counterions to balance the positive charge on the pyrrole functionalities; the Cl− ion can then be exchanged with anions that appear in the water that is being treated. Zhang et al. showed that PPy has a positive potential below pH 10, which could attach compounds with negative charges, such as the phosphate groups in a bacterial outer membrane [Polypyrrole (PPy) is a well-known semiconducting polymer that offers high electrical conductivity, environmental stability, and biocompatibility. Hong et al. deposited PPy into the pores of wood-based activated carbon for sulfate removal . Moreovemembrane .We synthesized an adsorbent by modifying GAC with PPy (PPy/GAC). The objectives of this work were to characterize the adsorbent and investigate its adsorption behaviors for MTBE. A biofilm reactor was established to achieve simultaneous adsorption and biodegradation of MTBE under continuous flow.The pyrrole monomer (98%) was purchased from Zhejiang Qingquan Pharmaceutical & Chemical Co., Ltd.  and distilled under vacuum. The distilled pyrrole was stored at 4 °C under the protection of nitrogen before use. Coconut shell-based GAC (40–80 mesh) was purchased from Sigma . Prior to use, the GAC was washed several times with deionized water to remove carbon fines, dried in an oven at 105 °C, and sterilized by autoclaving. MTBE was obtained from Sinopharm Chemical Reagent Co., Ltd. . Microbial consortium ERS, cultured in mineral salt medium (MSM) was used to inoculate the different adsorbent columns .3 as an oxidant using the soft-template method [3 solution  was slowly added to the mixture in a dropwise manner with stirring (30 min). The polymerization process continued for another 24 h. Finally, the resulting black precipitate was washed several times with deionized water until the filtrate became colorless and dried in a vacuum oven at 50 °C for 24 h.The PPy particles were synthesized in citric acid solution with FeCle method . The typ3 as the oxidant. First, the pre-treated GAC (2.5 g) was added to a pyrrole monomer solution (25 mL) at different concentrations , with magnetic stirring for 16 h. After filtration, a FeCl3 solution  was added dropwise to the formed suspension, and the mixed solution was stirred for another 6 h. Finally, the PPy/GAC composites were filtered and washed with deionized water. The prepared composites were dried at 50 °C for 24 h. The synthesized composites were named 0.1-PPy/GAC, 0.2-PPy/GAC, 0.5-PPy/GAC, and 1-PPy/GAC according to the concentration of the pyrrole monomer solution that was used during the preparation.The PPy/GAC composites were synthesized by the chemical oxidative polymerization of the pyrrole monomer (different concentrations) in the GAC suspension solutions with FeCl−1. The sample morphology was characterized by scanning electron microscopy . The Brunauer-Emmett-Teller (BET) surface area (SBET), total pore volume (V), and average pore radius (R) were measured at 77 K using a nitrogen adsorption apparatus .Fourier transform infrared (FT-IR) spectra of the samples were measured using the KBr pellet method with a TENSOR 37 FT-IR spectrophotometer  in the range of 4000–400 cmR (%), and the amount of dye molecules adsorbed on the adsorbents, tQ (mg/g), at certain times, t, were calculated from the following equations:C0 is the initial concentration of MTBE (mg/L), tC is the residual concentration at the special time point, t (mg/L), V is the solution volume (L), and M is the adsorbent mass (g).All adsorption experiments were performed in 50-mL serum bottles sealed with polytetrafluoroethylene (PTFE) stoppers at 30 °C. The suspension containing the 20 mL MTBE solution (1 mg/L) and 2 g/L of the adsorbent was stirred for 2 h then centrifuged at 4000 rpm for 5 min. The MTBE concentration in the supernatant was determined according to the following method. The effect of the dose of adsorbent on the MTBE adsorption was studied by adding different masses (0.5–10 g/L) of the adsorbent to a 1 mg/L MTBE solution. The adsorption rate, t is the adsorption time (min), K1 (min−1) and K2 (g/mg/min0.5) are the rate constants for the pseudo-first-order and pseudo-second-order models, respectively, and eqQ (mg/g) is the adsorption amount at the equilibrium state.The adsorption equilibrium of MTBE (1 mg/L) was evaluated at 25 °C. The adsorption kinetics was investigated using pseudo-first-order and pseudo-second-order models, according to the following equations, respectively:eqC (mg/L) is the MTBE equilibrium concentration, maxQ (mg/g) is the maximum adsorption capacity, LK (L/mg) is a constant that is related to the heat of adsorption, FK (mg/g) represents the adsorption capacity when eqC equals 1, and n represents the dependence of the degree of adsorption on the equilibrium concentration.Adsorption isotherms for MTBE on different adsorbents at 25 °C were obtained by mixing different concentrations of MTBE solutions (0.1–50 mg/L) with 2 g/L of the adsorbent. The Langmuir and Freundlich isotherms are described in linear forms according to Equations (5) and (6), respectively:600) of the circulating inoculum was used as an indication of biomass loading onto the adsorbent column. At three intervals over the four-week period, the spent medium was removed from the reactor and replaced with identical fresh medium supplemented with MTBE (1 mg/L) with an empty bed contact time (EBCT) of 28.5 min. In open mode operation, each column was continuously fed by fresh MSM  with MTBE at a concentration of 1 mg/L according to the conditions in Different adsorbents were packed into separate Plexiglass columns  at a bed height of 15 cm. A schematic of the column setup is presented in The bacterial communities attached to the different adsorbent columns were characterized by 16S-rRNA-based sequence analysis. Bacteria were recovered from the different adsorbents and were purified by repeated subculturing on nutrient agar and minimal salts agar supplemented with MTBE. Genomic DNA from a single strain was extracted using the Ezup Column Bacteria Genomic DNA Purification Kit  according to the manufacturer’s instructions. A partial 16S rRNA sequence was amplified from the genomic extraction with universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The amplification reaction mixtures (50 µL) contained 1 µL of the template, 1.5 U of PrimeSTAR HS DNA Polymerase , 10 µL of 5× PrimeSTAR buffer, 2 µL each of forward and reverse primers (10 µM), and 5 µL of a dNTP mixture (2.5 mM). Amplifications were performed using a T100 Thermal Cycler  at the following conditions: 98 °C for 5 min, 30 cycles at 98 °C for 10 s, 55 °C for 5 s, 72 °C for 90 s, and a final extension at 72 °C for 7 min. After purification, the polymerase chain reaction (PCR) product was Sanger-sequenced by Sangon Biotech .The DNA sequence was used to identify homologous sequences in GenBank with BLAST. Phylogenetic analysis of the 16S rRNA sequence was performed using the MEGA 5.1 software . Multiplxt autosampler  equipped with a polar, polyethylene glycol-coated WAX syringe, as described by Li et al. [MTBE concentrations were analyzed by headspace solid-phase dynamic extraction-gas chromatography-mass spectrometry (HS-SPDE-GC/MS). The liquid culture was collected and centrifuged at 6000 rpm for 10 min. Next, 2 mL of the supernatant was added to a 10-mL glass vial sealed with a screw cap and silicone-polyperfluoroethylene gaskets and immediately underwent HS-SPDE analysis on a CTC CombiPAL-i et al. . The samAll computer-based statistical analyses were performed with the SPSS 16.0 software for Windows . A one-way analysis of variance (one-way ANOVA) was used to assess the differences between treatments at a significance level of 0.01 or 0.05.−1 (quinonoid structure of PPy). The band at 1471 cm−1 is attributed to the C-N stretching vibration in the pyrrole ring. Additionally, the peaks at 1296, 1168, and 1032 cm−1 are ascribed to the in-plane -C-H vibrations, and the peak at 910 cm−1 is for the =C-H out-of-plane vibration [The FT-IR spectra  show theibration ,21. Thisibration .SBET), total pore volume (V), and average pore radius (R) of the different adsorbents varied with respect to the pyrrole monomer concentration (SBET = 58.67 m2/g) was extremely lower than that of the traditional GAC. Furthermore, both the BET surface area and the pore volume decreased with increasing pyrrole monomer concentration. These pore volume decreases confirmed that PPy successfully sorbed throughout the pore matrix of GAC; therefore, the FeCl3 catalyst likewise caused polymerization throughout this pore matrix. The decreased pyrrole monomer concentration enhanced the BET surface area, and the pore volume benefitted from the adsorption of MTBE. However, the reduced pore radius might have weakened the adsorption capacity of MTBE and the microbes.The specific surface area of the adsorbents, which is related to the adsorption properties, was analyzed. The BET surface area (ntration . The BETThe micropores (<20 Å) mostly consisted of the pore volume of GAC. Apart from possessing a high adsorption capacity, it is essential for an adsorbent to exhibit rapid adsorption kinetics for removing the adsorbate from the solution. R2 = 0.9856–0.9984). Furthermore, the calculated values of eqQ from the pseudo-second-order model were approximately equal to the experimentally obtained values. These results indicate that the adsorption kinetics of the synthesized adsorbents corresponded to the pseudo-second-order model.Moreover, the adsorption kinetics of MTBE was fitted by the pseudo-first-order and pseudo-second-order models. The related parameters for the adsorption kinetics were calculated and are listed in R2 is greater for the Langmuir model than that for the Freundlich model). The dimensionless separation factor, LR, which is an essential characteristic of the Langmuir model for reflecting the favorability of an adsorption process, is used:mC is the maximum initial concentration of MTBE in the solution. The LR values were calculated and are listed in LR for the four composites were in the range of 0–0.5, indicating that the adsorption processes were favorable.The adsorption isotherm was examined to determine the interaction between the adsorbate and adsorbent. The experimental parameters of the Freundlich and Langmuir isotherm models are listed in Based on the BET analysis, pyrrole monomer concentration, and MTBE-adsorption capacity of the four composites, 0.2-PPy/GAC was chosen for further analysis of the biofilm reactor.The microbial consortium ERS possesses a capacity for directly degrading MTBE . In thisAdsorption and biodegradation of organic compounds are factors that influence the performance of a column bioreactor. The reactor was operated as an open system (with no recirculation to vessel A) to quantify the degradation of MTBE by the column biofilm in continuous mode . The resIn this type of system , both the adsorption of target compounds to the adsorbents and biotreatment occur simultaneously. In this study, the MTBE that adsorbed to the adsorbents in the column slowly became bioavailable and was mineralized by the biofilm. A major advantage of MTBE adsorption in the column reactor was holding the compound for longer than the hydraulic retention time (HRT), allowing time for the biofilm consortium to effectively mineralize the compound. No MTBE was detected in the effluent of column 0.2-PPy/GAC at any time, even when the operation employed a low EBCT of 10 min . The MTBFor the reaction of biofilm column B with PPy, no MTBE was detected in the effluent during the first 12 days. However, after 12 days, a time breakthrough was observed in the effluent for the remainder of the experiment. The treatment performance was reduced with an increase of the feed rate using an EBCT of about 20 min (days 30–50). The EBCT was continuously decreased to 10 min, and the MTBE concentration in the effluent increased B. SimilaLee et al. reported that phenol and chlorinated phenols are more effectively biodegraded by immobilized bacteria than suspended bacteria . The remNo other studies have reported the treatment of MTBE-contaminated water with a microbial consortium attached to PPy-modified GAC. Thus, we needed to identify the microbial communities that were attached to the different adsorbents. At the end of the treatment, the development of the biofilm in the different adsorbent columns was monitored by SEM . MicrobiPseudomonas aeruginosa and Acinetobacter pittii were recovered from all three biofilms; they may be the predominant bacteria responsible for MTBE degradation in the biofilm reactors. Pseudomonas aeruginosa BM-B-450, which was isolated from an n-pentane-adapted consortium, can degrade MTBE [Pseudomonas genus have found innumerous environmental niches on Earth and have demonstrated the ability to mineralize various pollutants, including polycyclic aromatic hydrocarbons (PAHs) [Pseudomonas genus meet the requirements for industrial biotechnology, including high biomass production and low nutritional demands. Furthermore, numerous typical strains from the genus Pseudomonas have been whole-genome sequenced, including P. aeruginosa PAO1 [P. putida KT2440 [P. mendocina NK-01 [Pseudomonas genus strains in biotechnology and bioremediation. Acinetobacter, which occupies an important niche in nature, is another dominant genus that is ubiquitously distributed in numerous environments, including soil, sediments, water, and contaminated sites [Acinetobacter strains have the ability to mineralize versatile pollutants, such as various long-chain dicarboxylic acids, aromatic compounds, and hydroxylated aromatic compounds [Acinetobacter strains make them promising alternatives to Escherichia coli  for metabolism research. One of these desirable properties is the simplicity of genetic manipulation by homology-directed recombination with linear DNA fragments [Furthermore, a preliminary characterization of the biofilm microbial community was conducted. Several separate single strains were isolated from the biofilms on each column . A preliminary characterization of the recovered bacterial strains showed that all were gram-negative rods. The isolates were identified by 16S-rRNA-based phylogenetic analysis ; differeade MTBE . Memberss (PAHs) ,27, benzs (PAHs) , and alcs (PAHs) ,30. Straosa PAO1 , P. putia KT2440 , and P. na NK-01 . The broed sites ,35. Moreompounds ,37. The ragments .Sphingobium genus was only recovered from the 0.2-PPy/GAC column, Flavobacterium sp. was only isolated from the GAC column, Comamonas sp. was isolated from the 0.2-PPy/GAC and PPy columns, and Delftia sp. was isolated from the GAC and PPy columns. This suggests that biodegradation may be performed in a cooperative fashion by the biofilm bacteria, which may vary in composition accordingly to fluctuations in the feed. In addition, this may indicate that the typical degrading laboratory species may implant less successfully within treatment populations than naturally established bacterial communities in biofilms.The isolate belonging to the Pseudomonas sp. ) [Serratia sp., which accounted for 28.1% of the OTUs in ERS culture, had not been detected in the biofilm from all the three kinds of adsorbents. The decreased biodiversity in the mixed ERS after loaded on the carriers may be ascribed to the poor adhesion of some genera, e.g., Serratia sp., on the adsorbents.According to the reported results, the most dominant strains in ERS culture belong to  Units)) , which iPseudomonas aeruginosa and Acinetobacter pittii, which were recovered from all three biofilms, may be the predominant bacteria responsible for the observed MTBE degradation.In this study, GAC was successfully grafted with PPy by in-situ chemical oxidative polymerization of different concentrations of pyrrole monomer. The adsorption behaviors of MTBE on the composites were described by the pseudo-second-order and Langmuir isotherm models. The adsorption capacities decreased with the increasing concentrations of pyrrole monomer. In the subsequent biofilm reactor experiments, 0.2-PPy/GAC biofilm column exhibited a better and more enduring effect for the elimination of MTBE in continuous flow than PPy and GAC biofilm columns. Furthermore,